Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the central nervous system initiated by myelin antigen- specific encephalitogenic CD4+ Th1 cells. It has been suggested that recovery or protection from disease is mediated by Th2 cells. However, the critical cytokines responsible for the initiation and maintenance of disease, as well as those mediating recovery and relapse remain uncertain. During an immune response, the T cell requires 2 signals for full activation. The first signal is provided by engagement of the TCR with the antigenic peptide plus MHC molecule on antigen-presenting cells (ACPs), and the second "costimulatory" signal is provided by binding of specific receptors on T cells with their ligand/s on APCs. The best characterized costimulatory pathway is that provided by CD28 on T cells binding to B7-1 and B7-2 on professional APCs. Another costimulatory signal is provided by interaction of CD40 on the surface of APCs with CD40L on the surface of T cells. The role of specific costimulatory molecules in Th1 versus Th2 cell differentiation remains unclear. STAT4 knockout mice are unable to respond to IL-12 signaling and are thus unable to mount a Th1 response. STAT6 knockout mice are unable to respond to IL-4 and thus cannot mount a Th2 response. We have preliminary data using STAT 4 and STAT 6 knockout mice that suggest that Th1 and Th2 cells are differentially regulated by costimulatory activation during antigen priming. The purpose of this proposal is to dissect the interplay between specific T cell costimulatory activation pathways (CD28/CTLA4-B7 and CD40L-CD40) and Th1 and Th2 cytokines in regulating immune responses in vivo in a clinically relevant autoimmune disease model characterized by remissions and relapses. First, we will study the role of Th1 and Th2 cytokines in initiating disease, effecting recovery, or causing relapses using STAT 4 and STAT 6 knockout mice which have been extensively backcrossed unto EAE susceptible strains. We will also use MBP TCR transgenic mice intercrossed with STAT KO mice to address mechanisms. Second, we will study mechanisms of costimulatory signal (CD28, CD40L, and CTLA4) blockade in STAT 4 and STAT 6 knockout mice. We will also determine the differential effects of blocking CD28-B7 or CD40L-CD40 in the generation of a Th1 or Th2 response in vivo. These studies should have relevant clinical implications for autoimmune diseases such as multiple sclerosis, and may provide the rationale for development of novel therapies targeted at blocking T cell costimulation in immunologically mediated diseases.